CD8+ T cells recognize class I molecules of the major histocompatibility complex (MHC) bearing peptides of 8 to 10 residues derived from cytosolic proteins. These cells are a bulwark of host defenses to infectious agents and tumors, and it is critical to understand how antigenic peptides are generated by cells if we are to improve existing vaccines and develop new vaccines and treatments for infectious and neoplastic diseases. Very little is known about how cells produce antigenic peptides from proteins. While it is clear that proteolytic production of peptides begins in the cytosol, it is uncertain to what extent trimming occurs after peptides are translocated into the endoplasmic reticulum (ER). Previous results from our laboratory indicate that trimming of peptides in the ER can occur under the special circumstances of expressing an exotic secretory protease in antigen-presenting cells. To extend these findings, we have used a novel strategy to characterize the endogenous proteolytic capacity of the ER and its relevance to antigen processing. Our findings demonstrate that ER proteases, most likely aminopeptidases, are able to liberate antigenic peptides from longer precursors. In conjunction with recent findings from other laboratories, our results suggest a model in which peptides from cytosolic proteins are transported into the ER with amino terminal extensions that are removed by an aminopeptidase residing in the ER.